VCF preprocessing

We used a VCF file created on the basis of snarl traversal of the MC graph as a basis for genotyping. The records contained in this VCF represent bubbles in the underlying pangenome graph and their nested variants, derived from the snarl tree. Each variant was marked according to their level in this tree. Variants annotated by ‘LV=0’ correspond to the top-level bubbles. We used vcfbub (v.0.1.0)¹⁰⁰ with parameters -l 0 and -r 100000 to filter the VCF. This removed all non-top-level bubbles from the VCF unless they were nested inside a top-level bubble with a reference length exceeding 100 kb; that is, top-level bubbles longer than that are replaced by their child nodes in the snarl tree. The VCF also contained the haplotypes for all 44 assembly samples, representing paths in the pangenome graph. We additionally removed all records for which more than 20% of all 88 haplotypes carried a missing allele (“.”). This resulted in a set of 22,133,782 bubbles. In a next step, we used PanGenie (v.1.0.0)⁵⁴ to genotype these bubbles across all 3,202 samples from the 1KG based on high-coverage Illumina reads¹⁹.